Replaceable emitter assembly for interfacing a separation column to a mass spectrometer

ABSTRACT

An electrospray emitter assembly for interfacing a separation column to a mass spectrometer is disclosed. An emitter capillary includes an inlet end and an outlet end. A fitting is coupled to the inlet end of the emitter, configured to be removably connected to the separation column. A stop with a defined through hole is integrated proximate the inlet end of the emitter to produce a path for liquid to flow from the separation column to the emitter via the through hole where a voltage is applied to the liquid entering the emitter.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation under 35 U.S.C. § 120 andclaims the priority benefit of co-pending U.S. patent application Ser.No. 16/852,769, filed Apr. 20, 2020, which is a continuation of U.S.patent application Ser. No. 15/649,220, filed Jul. 13, 2017, now U.S.Pat. No. 10,627,375, which claims the benefit of U.S. ProvisionalApplication No. 62/361,692, filed Jul. 13, 2016. The disclosures of eachof the foregoing applications are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to electrospray emitter capillaries. Morespecifically, this invention relates to a low dead volume replaceableemitter assembly for interfacing a separation column to a massspectrometer.

BACKGROUND OF THE INVENTION

Mass spectrometry (MS) has become an essential analytical tool due toits high sensitivity and ability to detect and identify a large numberof analytes while also providing structural information of variousmolecules, which is very useful especially for biological sampleanalysis. Liquid chromatography (LC) is the advanced separationtechnique to resolve different species dissolved in the liquid phase,principally based on the difference of molecular properties.

LC-MS, a combination of powerful separation and identificationtechniques, is currently widely used in a broad range of fields,including pharma, biopharma, clinical, environmental, food safety,forensic, omics (proteomics, metabolomics, genomics, glycomics, and soon), academic research, regulatory agents, etc. As the front end tool,LC separates the components present in the sample and/or reduces thesample complexity before entering into MS for detection.

Electrospray ionization (ESI) is a technique to produce gas phase ionsfrom liquid solution while applying a high voltage to create aerosol.ESI is commonly used as the interface to couple liquid phase separationmethods (LC and CE) with MS. The ESI emitter, typically a needle orcapillary made from fused silica, metal, or glass, connects with the LCcolumn outlet and is located in the front of MS. As a soft ion source,ESI can create multiply charged molecule ions from LC elution, whichprovide the molecular weight information of the analytes of interest andare prone to conduct tandem MS (e.g., MS²) to explore their structureinformation.

ESI interfaces play an important role on LC-MS sensitivity. It is wellknown that ion loss during electrospray is one of the major causesaffecting MS detection sensitivity. ESI efficiency or ionizationefficiency increases with the decrease of the liquid flow rate and canapproach 100% at low nL/min flow rates [Smith, R. D.; Shen, Y.; Tang, K.Acc. Chem. Res. 2004, 37, 269-278]. Very often, limited sample amounts,sample complexity, and the quest for highest possible MS sensitivityrequire the use of small inner diameter columns from 1.0-mm down to 20μm, with corresponding operating flow rates from 100 μL/min to less than20 nL/min. With a decrease in column inner diameter, the operating flowrate also decreases, which is more suitable for high sensitive MSdetection.

An ESI interface also affects the LC separation performance because itintroduces the post-separation extra-column dwell volume (dead volume)due to required connections, which will broaden the peaks and,therefore, decrease the resolution, signal intensity, and also impact MSdetection (e.g. data-dependent MS²). Minimizing such extra column dwellvolume is essential to achieve desired LC separation and MS results.Although small transfer lines are used to connect the column and emitterwhen using traditional ESI source, the presence of too many connectionsand transfer lines still affects the quality of data. This impactbecomes more significant for low flow columns, particularly nano columns

Typically the nano spray emitter is directly attached to the nano columnoutlet using a union to minimize the extra-column dwell volume (no extratransfer line and few connections). Currently, most end-users assemblethe nano spray emitter with the nano LC column by themselves using thesleeves and fittings. However, this approach is prone to improperconnections resulting in leaks, column/emitter breaks, or undesireddead-volumes, and consequently poor chromatographic separation anddetection sensitivity. Also it may be difficult to achieve results withgood reproducibility due to large variation of the emitter dimension andconnections. The plug unit and connection system disclosed in US2015/9091693 (e.g. nanoViper™ fitting, Thermo Fisher Scientific,Waltham, Mass., USA) has a virtually zero dead volume for connectingcapillary tubes. It is convenient to achieve reliable connections,especially for ultra-high pressure liquid chromatography.

Besides selecting the correct LC column for desired separations, e.g. anano LC column, minimizing, even eliminating, user intervention with theanalytical techniques is also demanded to ensure reproducible results. Amicrochip based LC-ESI device integrates a trapping column, a separationcolumn, and an electrospray emitter within a single structure [Fortier,M. H.; Bonneil, E.; Goodley, P.; Thibault, P. Anal. Chem. 2005, 77,1631-1640]. While this integrated system has less dead volume, thechromatographic performance is currently not able to compete with thatof conventional non-chip based systems due to the technology limitationsand low column pressure rating.

The EASY-Spray™ column (Thermo Fisher Scientific, Waltham, Mass., USA)is an integrated system mainly containing a separation column andheating unit embedded in a plastic material, and an electrospray emitterwhich is connected with the separation column and protected by aretractable sleeve. [WO 2013/167131]. Because the connection of thecolumn and emitter is embedded in the plastic material, the emitter isnot able to be replaced when it fails, for example, due to clogging. Inthis case, the entire system is unusable even though the separationcolumn is still functional.

To overcome the above challenges, it is desired to develop a stand-aloneemitter assembly that provides a low dead-volume connection between theseparation column and the mass spectrometer (or ion source of the massspectrometer), is compatible with a broad range of flow rates, includingnano LC-MS applications, and is easily replaced.

SUMMARY

Embodiments of the present invention disclose an electrospray emitterassembly for interfacing a separation column to a mass spectrometer. Inone embodiment of the present invention, the emitter assembly comprisesan emitter capillary having an inlet end and an outlet end. A fitting iscoupled to the inlet end of the emitter, and the emitter is configuredto be removably connected to the separation column via the fitting. Astop with a defined through hole is integrated proximate the inlet endof the emitter to produce a path for liquid to flow from the separationcolumn to the emitter via the through hole. A voltage is applied acrossthe stop with the through hole to the liquid entering the emitter.

The fitting may be a female threaded end fitting configured forengagement with a plug type capillary fitting of the separation column.

The emitter may comprise, but is not limited to, a fused silicacapillary, a metal capillary, a ceramic capillary, or a glass capillary.

In some embodiments, the separation column is removably connected via atransfer line or capillary with the emitter capillary through thefitting. The separation column may be a liquid chromatography (LC)column.

In some embodiments, the integrated stop has a thickness of up to 1.0 mmIn some other embodiments, the integrated stop has a thickness ofbetween 100 μm to 300 μm.

In some embodiments, the through hole in the stop has a diameter ofbetween 3 μm and 100 μm.

The emitter assembly may include a retractable protective sleeve forcovering and supporting the emitter. In some embodiments, theretractable protective sleeve is slidably mounted around the emitter andmoveable to an extended position where the tip of the emitter is coveredby the protective sleeve. A resilient member, such as a spring, isprovided to bias the protective sleeve towards the extended positioncovering the tip of the emitter. The retractable protective sleeve isalso moveable to a retracted position such that the tip is uncovered.

The emitter assembly may further include an electrically conductiveouter sheath, such that the protective sleeve is enclosed and moveablewithin the outer sheath.

In another embodiment of the present invention, an electrospray emitterfor interfacing a separation column to a mass spectrometer is disclosed.The emitter assembly includes an emitter capillary having an inlet endand an outlet end. A plug type end fitting is coupled to the inlet endof the emitter, and the emitter is configured to be removably connectedto the separation column via the plug type end fitting. In someembodiments, the plug type end fitting is a male threaded end fitting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of cross-sectional side view of theemitter assembly with a female end fitting, in accordance with oneembodiment of the present invention.

FIG. 2 is an outside view of the emitter assembly of FIG. 1.

FIG. 3 is a schematic diagram of a cross-sectional side view of theemitter assembly of FIG. 1 inserted in a holder of a mass spectrometerinstrument, in accordance with one embodiment of the present invention.

FIG. 4 is a schematic diagram of a cut out section of the emitterassembly of FIG. 1, focusing on the stop integrated into the emitterassembly.

FIG. 5 shows the emitter assembly in a pinning fixture before theemitter body is pinned to the union.

FIG. 6 shows the emitter assembly in a pinning fixture as the pins areactively connecting the emitter body and the emitter union.

FIG. 7 is a schematic diagram of cross-sectional side view of theemitter assembly with a plug type end fitting, in accordance with oneembodiment of the present invention.

FIG. 8 is an outside view of the emitter assembly of FIG. 7.

FIG. 9 is a schematic diagram of a cross-sectional side view of theemitter assembly of FIG. 7 inserted in a holder of a mass spectrometerinstrument, in accordance with one embodiment of the present invention.

FIG. 10 is a close up view of part of the emitter assembly of FIG. 1.

FIG. 11 is an example of an emitter assembly, similar to FIG. 1, coupledwith a nano- or capillary column, in accordance with one embodiment ofthe present invention.

FIG. 12 is a close view of part of the emitter assembly of FIG. 7.

FIG. 13 is an example of an emitter assembly, similar to FIG. 7, coupledwith a capillary column, in accordance with one embodiment of thepresent invention.

FIG. 14 is an example of an emitter assembly, similar to FIG. 7, coupledwith a micro- or analytical column, in accordance with one embodiment ofthe present invention.

FIG. 15 shows a base peak chromatogram of the BSA tryptic digestanalyzed using a 50 μm inner diameter column coupled with the femaletype emitter assembly described in Example 1.

FIG. 16 shows the extracted ion chromatogram of ion with m/z 722.

FIG. 17 shows a base peak chromatogram of the BSA tryptic digestanalyzed using a 75 μm inner diameter column coupled with the femaletype emitter assembly described in Example 1.

FIG. 18 shows the extracted ion chromatogram of ion with m/z 722.

FIG. 19 shows a base peak chromatogram of the BSA tryptic digestanalyzed using a 150 μm inner diameter column coupled with the femaletype emitter assembly described in Example 2.

FIG. 20 shows the extracted ion chromatogram of ion with m/z 722.

FIG. 21 shows a base peak chromatogram of the BSA tryptic digestanalyzed using a 150 μm inner diameter column coupled with the male typeemitter assembly described in Example 3.

FIG. 22 shows the extracted ion chromatogram of ion with m/z 722.

FIG. 23 shows a base peak chromatogram of the BSA tryptic digestanalyzed using a 250 μm inner diameter column coupled with the male typeemitter assembly described in Example 3.

FIG. 24 shows the extracted ion chromatogram of ion with m/z 722.

FIG. 25 shows a base peak chromatogram of the BSA tryptic digestanalyzed using a 500 μm inner diameter column coupled with the male typeemitter assembly described in Example 4.

FIG. 26 shows the extracted ion chromatogram of ion with m/z 722.

FIG. 27 is a column performance comparison between the assembly used inExample 5 and a fully integrated EASY-Spray™ column (ES801, ThermoFisher Scientific), showing the base peak chromatograms of theseparation and the extracted ion chromatograms of ion with m/z 722 fromboth columns.

FIG. 28 is a column performance comparison between the assembly used inExample 6 and a fully integrated EASY-Spray™ column (ES804, ThermoFisher Scientific), showing the base peak chromatograms of theseparation and the extracted ion chromatograms of ion with m/z 722 fromboth columns.

FIG. 29 is a column performance comparison between the assembly used inExample 7 and a fully integrated EASY-Spray™ column (ES806, ThermoFisher Scientific), showing the base peak chromatograms of theseparation and the extracted ion chromatograms of ion with m/z 722 fromboth columns.

DETAILED DESCRIPTION OF EMBODIMENTS

FIG. 1 is a schematic diagram of cross-sectional side view of theemitter assembly with a female end fitting, in accordance with oneembodiment of the present invention. The embodiment shown in FIG. 1includes an electrospray emitter 130 held in place with PEEK sleeve 135,cap nut 170 and ferrule 180. The emitter is typically a fused silica,metal, glass, or ceramic needle or capillary as known in the LCMScommunity. The fitting for the emitter is a conventional type forcoupling capillary tubing. A LC column with a threaded male end can beconnected to the threaded female inlet on the opposite side of theemitter in union 120.

At or near the inlet of the emitter 130, a stop 100 is integrated intothe union 120 with a defined through hole to ensure a proper voltageapplication to the liquid entering the emitter. The other side of theunion 120 is a fitting for receiving a number of standard capillaryconnections. The union 120 includes an externally threaded side 133 anda threaded inlet side 122.

A protective sleeve 140 of generally cylindrical form is slidablylocated on the emitter 130. The sleeve 140 has a main body 110 and abase 111 of a wider diameter than the main body. The protective sleeve140 is generally made of plastic. A PEEK sleeve 135 covers at least acentral portion of the emitter 130 and is adapted to closely fit betweenan outer diameter of the emitter 130 and the protective sleeve 140.Mounted around the protective sleeve 140, in one embodiment, is anelectrically conductive sheath 150. The conductive sheath is supportedat one end by the cap nut 170. The sheath may be detached from thecolumn fittings at that end. The conductive sheath 150 has an internaldiameter such as to accommodate therein the protective sleeve 140 andpermit the protective sleeve 140 to slidably move in a reciprocatingmanner inside the sheath, described in further detail below.

A resilient member or spring 160 is provided inside the electricallyconductive sheath 150, positioned in a space between the emitterfittings and the protective sleeve 140, thereby to act upon the base ofthe protective sleeve. In this way, the spring 160 biases the sleeve 140to force it out of the conductive sheath 150. The length of the sleeve140 and its extension out of the sheath is sufficient to cover the tipof the emitter 130 and act to protect it against damage. A part of themain body 110 of the protective sleeve 140 protrudes outside the sheath150 and thereby covers the emitter. The extent of travel of the sleeve140 out of the sheath 150 is restricted by a reduced internal diameterpart 190 at the end of the sheath 150 that stops the wider diameter base111 of the sleeve. If a force is applied to the sleeve to push thesleeve backwards into the sheath 150 the spring 160 becomes compressedand the tip of the emitter becomes exposed and ready for use. Theelectrically conductive sheath 150 has a recess in the form of acircumferential groove 149 in its outer surface for the purpose ofmaking contact with a high voltage contact, e.g. a contact ball, asdescribed further below.

In some embodiments, the protective sleeve is fixed with respect to theemitter. However the protective sleeve may also be retractable withrespect to the emitter. Where the sleeve is retractable, this ensuresthat the emitter tip is exposed when in use and thereby the sleeve doesnot interfere, for example, with gas flows and equipotential linesaround the emitter tip. Moreover, a retractable sleeve, when in use,does not block visibility of the emitter so one can readily monitor thespray. In some embodiments, the protective sleeve is slidably located onthe emitter. In this case, the protective sleeve is movable between anextended (or cover) position wherein it covers the emitter, especiallythe tip thereof, and a retracted position wherein the emitter,especially the tip thereof, is exposed. When the emitter is exposed itmay be used for electrospray ionization. The emitter tip herein meansthe tip from which ions are produced when in use. The protective sleevethus covers and supports the electrospray emitter along at least aportion of its axis which includes the tip of the emitter.

In some embodiments, the protective sleeve comprises a generallycylindrical body that surrounds and supports the emitter. The generallycylindrical body may comprise a base of greater diameter than aremainder or main body of the sleeve. The resilient member (e.g., aspring) is provided in contact with the protective sleeve, to bias thesleeve towards its extended position. The resilient member is in contactwith a base of the protective sleeve and positioned between a connectionfitting and the protective sleeve. In this way, the resilient member,upon activation, is able to force the sleeve to cover the emitter whenit is required to be protected. The resilient member also allows thesleeve to be retracted from the tip end of the emitter when the emitteris required to be used, e.g. when the emitter assembly is assembled withan instrument for mass spectrometric analysis. To enable thisretraction, the resilient member is forced into a compressed state, e.g.by pushing the sleeve towards the resilient member. The resilient memberbiases the sleeve to the extended position such that the sleeve adoptsthe extended or cover position when the sleeve does not have asufficient force applied pushing it against the resilient member. Theresilient member or spring thereby enables the protective sleeve tocover the tip end of the emitter when the emitter is not required to beused, e.g. when the emitter assembly is disassembled from an instrumentfor mass spectrometric analysis.

The protective sleeve may be enclosed within an outer sheath, which incertain aspects of the invention is the electrically conductive sheathdescribed herein. The outer sheath may be fixed in position in relationto the emitter. The protective sleeve is capable of reciprocating motionwithin the outer sheath, thereby enabling the protective sleeve to beretractable with respect to the emitter. In such embodiments, theresilient member is also provided inside the outer sheath for providinga force against the sleeve, or against the base of the sleeve, to biasthe sleeve towards the extended position.

Thus, in embodiments where the protective sleeve is used in combinationwith the electrically conductive sheath, the resilient member isprovided inside the electrically conductive sheath between the fittingand the protective sleeve covering, whereby the spring, upon activation,is able to force the sleeve out of the sheath to cover the emitter.Thus, in certain embodiments, the protective sleeve may be forced out assoon as the system is pulled out of a recipient holder (as described inmore detail below), i.e. the spring force is constantly acting so as topush the sleeve in an outwards direction thereby to cover the emitter.In some embodiments, the protective sleeve is made of a rigid materialsuch as a metal or a polymer material. In this way the rigidity of thesleeve can protect the fragile emitter that it covers.

In some embodiments, the separation column is connected via a transferline or capillary with the electrospray emitter through one or more endfittings. It should be noted that the separation column may be directlycoupled or connected with the replaceable emitter through the endfitting, without using a transfer line. The overall design may thereforebe made as a convenient “connect-and spray” type, which the user onlyhas to connect the separation column with a finger tight fitting and fitinto a receiving frame or holder on an instrument, e.g. for massspectrometry.

FIG. 2 is an outside view of the emitter assembly of FIG. 1, showing theelectrically conductive sheath mounted at the front end and theprotruding sleeve protecting the emitter (not visible). It will beappreciated from the description that the emitter assembly system may beformed as a type of cartridge for use with a LC separation column and aninstrument, e.g. mass spectrometer.

FIG. 3 is a schematic diagram of a cross-sectional side view of theemitter assembly of FIG. 1 inserted in a holder of a mass spectrometerinstrument, in accordance with one embodiment of the present invention.As shown in FIG. 3, a holder 166 or adaptor is shown that fits to theouter shape of the electrically conductive sheath 150. The holder 166may be fixed on the laboratory instrument (e.g. mass spectrometer) thatis not shown in the figure. The electrically conductive sheath 150 hasan outer shape and provides a close, tight, fit in the receiving holder166. In this specific embodiment, the electrically conductive sheath 150has a circular cylindrical outer shape and the holder 166 has a circularcylindrical receiving space to receive the electrically conductivesheath 150.

The electrically conductive sheath may be enclosed within a holderhaving a high voltage contact point when the emitter assembly is in use.The holder may be located or positioned on an instrument, e.g. for massspectrometric analysis. The high-voltage contact point may be anelectrically conductive ball 155 fitting a recess, such as a groove 124,in the outer surface of the electrically conductive sheath. The groove124 may be a circumferential groove in the outer surface of theelectrically conductive sheath. The contact point may be, for example, aspring loaded ball bearing. The electrically conductive sheath may havea shape that provides a close or tight fit in a receiving holder on alaboratory apparatus (e.g. mass spectrometer). In a specific embodiment,the electrically conductive sheath has a cylindrical, i.e. circularcylindrical, outer shape. In that embodiment, the holder has acylindrical, i.e. circular cylindrical, receiving space, to receive theelectrically conductive sheath of the emitter assembly. In this way,with such cylindrical shaped parts and a groove in the outer surface ofthe electrically conductive sheath to receive the high voltage contactpoint, an exact angular alignment of the emitter assembly in the holderis optional. An axial alignment of the emitter assembly in the holdercan be achieved by means of providing an appropriate stop within theholder and/or by means of the contact point fitting in the groove in theouter surface of the electrically conductive sheath.

The ball 155 for the high voltage (HV) connection, which also provides asnap-on connection for the electrically conductive sheath 150, can be aspring loaded ball bearing that fits the groove 124 in the outer surfaceof the electrically conductive sheath 150 and provides the HV connectionthere to. Axial alignment of the emitter assembly in the holder 166 isachieved by means of inserting the electrically conductive sheath 150 ofthe emitter assembly into the holder 166 until the HV ball contact 155fits in the grove 124 in the outer surface of the electricallyconductive sheath and snaps or locks into the assembly into place withinthe holder 166. The female fitting electrically contacts theelectrically conductive liquid (eluent) at the point of entering thethrough a hole in the stop 100 (FIG. 1) between the emitter inlet andexit of the LC column enabling the transfer of charge from the highvoltage contact point to the tip of the emitter.

The emitter assembly also permits simultaneous retraction of theprotective sleeve 140 from the emitter tip as the emitter assembly isinserted in the holder 166 to allow the emitter to be used, e.g. in themass spectrometer. For this purpose, while the emitter 130 and the PEEKsleeve 135 fit through an orifice 179 in the holder 166, the main body110 of the sleeve 140 does not. The orifice 179 may lead into anionization chamber, e.g. of a mass spectrometer. In this embodiment, thesleeve 140 has an end portion of reduced diameter 187 compared to itsmain body 110. In this way, the reduced diameter portion 187 of theprotective sleeve fits through the orifice in the holder 166 and maythereby support the emitter in this region.

It will be appreciated that when the integrated assembly is withdrawnfrom the holder 166, movement of the sleeve 140 will no longer berestricted by the wall surrounding the orifice 179 in the holder 166such that the spring 160 will force the sleeve 140 out of the sheath 150once again so as to cover and protect the emitter 130 as shown in FIG.1.

FIG. 4 is a schematic diagram of a cut out section of the emitterassembly of FIG. 1, focusing on the stop integrated into the emitterassembly. The threaded inlet side 122 of the union 120 allows for astandard capillary connection to be pressed against the stop 100. Thethread-less side 133 of the union 120 allows for the emitter 130 to beconnected to the outlet side of the stop 100. The emitter is held inplace with cap nut 170 and ferrule 180. The defined through hole 144 inthe stop 100 allows for the liquid (e.g., from the separation column) tocome into contact with the union 120 and completes the electrical pathfrom the HV ball contact 155 to the tip of the emitter 130. This pathwayensures that there is no loss in electrical application negativelyaffecting the electro spray.

FIG. 5 shows the emitter assembly in a pinning fixture before theemitter body is pinned to the union. FIG. 6 shows the emitter assemblyin a pinning fixture as the pins are actively connecting the emitterbody and the emitter union.

The pins, which may be activated by compressed air, are pressed againsta wall of the body where the indentations or compression points arelocated. After the pins press against these spots and deform the body,the pins retract to the starting position. The assembly is rotatedapproximately 90° and the same action occurs. In one embodiment, thepins are not added to the assembly and the deformation binds 150(FIGS. 1) and 170 (FIG. 1) together.

Fittings as used for the present invention may be any fittings for LC.The fittings may be constructed from a wide range of polymer materialsor made from a range of metals. For the purpose of making electricalcontact between an electrospray potential as described herein, it isadvantageous that the fitting materials be conductive.

Fittings may include ferrules or gaskets that provide a seal between thebody of the fitting and the conduits that are to be connected. Tosolidify the electrical connection between the outlet of the LC columnto the inlet of the emitter, the fitting contains an integrated stop inthe union. The union is comprised of a conductive material such asstainless steel to ensure a good electrical path through the union tothe liquid or eluent. The thickness of the integrated stop is from 0 to1 mm with a preferred thickness of between 100 μm to 300 μm. A hole isplaced in the stop to allow a path for the liquid to flow from the exitof the LC column to the inlet of the emitter. The diameter of this holein the stop is between 1 μm and the outside diameter of the sleevecovering the emitter with a preferred diameter of 3 μm to 100 μm. Insome embodiments, the through hole is disposed at or near the middle ofthe stop. However, the through hole may be disposed at any portion ofthe stop.

The electrospray emitter as used for the present invention may be of anyconstruction. The electrospray emitter may be made of fused silica,metal, glass, or ceramic tubing, which may end in a sharp or blunt tip.It is usually preferable to have a sharp and tapered tip such as thatobtained when using an automated capillary puller (e.g. from Sutterinstrument, Inc., Novato, Calif., USA) since such emitters provide amore stable spray than blunt emitters do. Typically such taperedemitters have an outer diameter of about 360 μm and an inner diameter of5 μm to 100 μm whereas the orifice at the tapered tip is usually around1 μm to 20 μm. The length of such emitters is usually between 30 mm and60 mm but may also be longer or shorter.

The electrospray emitter assembly allows a user to connect the emitterto any standard fitting such as, but not limited to, a 10/32 fitting forUHPLC. The electrospray emitter utilizes the retractable protectivesleeve for covering and supporting the electrospray emitter along aportion of its access. Attached to the end of the emitter is a plug unitand connection system for connecting capillary tubes for highperformance liquid chromatography.

Some embodiments of the present invention use stainless steel emittersthat have an outer diameter between 100 μm and 500 μm and an innerdiameter between 5 μm and 100 μm. Some embodiments of the presentinvention use polished fused silica glass emitters that have an outerdiameter between 100 μm and 500 μm and an inner diameter between 2 μmand 100 μm.

FIG. 7 is a schematic diagram of cross-sectional side view of theemitter assembly with a plug type end fitting, in accordance with oneembodiment of the present invention. The arrangement shown in FIG. 7comprises an electrospray emitter 130 inserted or threaded through thePEEK sleeve 135 of the emitter 130 and held in place with ferrule 177and metal sleeve 220. PEEK sleeve 221, together with metal sleeve 220,facilitates connection of a fitting, such as a nanoViper™ fitting, at aninlet of the emitter 130. The emitter may comprise a fused silica,metal, ceramic, or glass needle or capillary as known in the LCMScommunity. The emitter is held in place with a plug type capillaryfitting and can be connected to a LC column by a threaded or press fit.

A protective sleeve 140 of generally cylindrical form is slidablylocated on the emitter 130 in the PEEK sleeve 135. The protective sleeve140 has a main body 110 and a base 111 of a wider diameter than the mainbody. The protective sleeve 140 is generally made of plastic. Mountedaround the protective sleeve 140, in some embodiments, is anelectrically conductive sheath 150 made of metal. The conductive sheathis supported at one end by the threaded fitting 211 which is made ofmetal. The sheath 150 may be detached from the column fittings at thatend. The conductive sheath 150 has an internal diameter such as toaccommodate therein the protective sleeve 140 and permit the protectivesleeve 140 to slidably move in a reciprocating manner inside the sheathas described below.

A spring 160 is provided inside the electrically conductive sheath 150,positioned in a space between the emitter fittings and the protectivesleeve 140, thereby to act upon the base of the protective sleeve 140.In this way, the spring 160, biases the sleeve 140 to force it out ofthe conductive sheath 150. The length of the sleeve 140 and itsextension out of the sheath is sufficient to cover the tip of theemitter 130 and act to protect it against damage. A part of the mainbody 110 of the protective sleeve 140 protrudes outside the sheath 150and thereby covers the emitter. The extent of travel or movement of thesleeve 140 out of the sheath 150 is restricted by a reduced internaldiameter part 190 at the end of the sheath 150 that stops the widerdiameter base 111 of the sleeve. When a force is applied to the sleeveto push the sleeve backwards into the sheath 150 the spring 160 becomescompressed and the tip of the emitter becomes exposed and ready for useas described in more detail below.

The electrically conductive sheath 150 has a recess in the form of acircumferential groove 149 in its outer surface for the purpose ofmaking contact with a high voltage contact, e.g. a contact ball, asdescribed further below.

FIG. 8 shows the arrangement of FIG. 7 from the outside showing theelectrically conductive sheath mounted at the front end and theprotruding sleeve protecting the emitter (not visible). It will beappreciated from the description that the whole emitter assembly is thusformed as a type of cartridge for use with a LC separation column and aninstrument, e.g. mass spectrometer.

FIG. 9 is a schematic diagram of a cross-sectional side view of theemitter assembly of FIG. 7 inserted in a holder of a mass spectrometerinstrument, in accordance with one embodiment of the present invention.As shown in the figure, a holder 166 or adaptor is shown that fits tothe outer shape of the electrically conductive sheath 150 which enclosesthe protective sleeve 140 and provides an electrical connection. Theholder 166 may be fixed on the laboratory instrument (e.g. massspectrometer) that is not shown in the figure. The electricallyconductive sheath 150 has an outer shape and provides a close, tight,fit in the receiving holder 166. In this specific embodiment, theelectrically conductive sheath 150 has a circular cylindrical outershape and the holder 166 has a circular cylindrical receiving space toreceive the electrically conductive sheath 150.

A ball 155 for the high voltage (HV) connection, which also provides asnap-on connection for the electrically conductive sheath 150, is alsoshown. The ball 155 may be a spring loaded ball bearing that fits thegroove 124 in the outer surface of the electrically conductive sheath150 and provides the HV connection there to. Axial alignment of theemitter assembly in the holder 155 may be achieved by means of insertingthe electrically conductive sheath 150 of the emitter assembly into theholder 166 until the HV ball contact 155 fits in the grove 124 in theouter surface of the electrically conductive sheath and snaps or locksinto the assembly into place within the holder 166. The electricallyconductive sheath 150 encloses and electrically contacts the threadedfitting 211 that, along with the metal sleeve fitting 220 and PEEKsleeve 221, connects the emitter to an upstream LC column The fittingelectrically contacts the emitter with the fitting on the outlet of theLC column. As the liquid (eluent) exits the LC column it is electricallycharged as it enters the electrospray emitter 130.

The emitter assembly also permits simultaneous retraction of theprotective sleeve 140 from the emitter tip as the emitter assembly isinserted in the holder 166 to allow the emitter to be used, e.g. in themass spectrometer. For this purpose, while the emitter 130 fits throughan orifice 179 in the holder 166, the main body 110 of the sleeve 140does not. The orifice 179 may lead into an ionization chamber, e.g. of amass spectrometer. In this embodiment, the sleeve 140 has an end portion187 (FIG. 3) of reduced diameter compared to its main body 110. In thisway, the reduced diameter portion 187 of the protective sleeve fitsthrough the orifice 179 in the holder 166 and may thereby support theemitter in this region.

It will be appreciated that when the emitter assembly is withdrawn fromthe holder, the travel or movement of the sleeve 140 will no longer berestricted by a wall surrounding the orifice 179 in the holder 166 suchthat the spring 160 will force the sleeve 140 out of the sheath 150 onceagain so as to cover and protect the emitter 130 as shown in FIG. 7.

EXAMPLE 1 Assemble the Female Type Emitter Assembly with 7 μm ID Emitter

An integrated emitter assembly having the structure shown in FIG. 1 wasassembled, which has a female type end fitting. The emitter was madefrom a fused silica capillary with 7 μm ID and 150 μm OD. The emitteroutlet was polished to a sharp point. The emitter length is 30 mm Thefemale type end fitting is compatible with the plug type capillaryfitting. The female union has a 250 um stop with a through hole of 50 μmID between the emitter entrance and the plug type capillary fitting (SeeFIG. 10, which is a close up view of part of the emitter assembly ofFIG. 1, and table 1 for a description of the union and emitter). FIG. 11is an example of the assembled emitter and nano/capillary column, inaccordance with one embodiment of the present invention.

TABLE 1 Details for FIG.10 in Example 1. Element Number in FIG. 10Dimensions 100 Length: 0.25 mm 144 ID: 0.05 120 10/32″ Threading,Length: 8 mm 180 PEEK ferrule 130 Fused Silica, ID: 7 μm, OD: 150 μm,Length: 30 mm 133 Ferrule Seat in Union, 8/32″ Threading 135 PEEKtubing, OD 1/32″, ID 180 μm, Length 26 mm

The replaceable emitter assembly was assembled by first inserting theemitter into the protective PEEK sleeve. The nut and ferrule were thenplace over the PEEK sleeve and inserted into the male end of the union.The ferrule was set into the union to prevent the emitter from moving.The emitter was then threaded through the spring, sliding emitterprotector and emitter casing. The casing is then attached to the nut andunion by pressing on the pin points of the casing.

EXAMPLE 2 Assemble the Female Type Emitter Assembly with 15 μm IDEmitter

An integrated emitter assembly having the structure shown in FIG. 1 wasassembled, which has a female type end fitting. The emitter was madefrom a fused silica capillary with 15 μm ID and 150 μm OD. The emitteroutlet was polished to a sharp tip. The emitter length is 30 mm Thefemale type end fitting is compatible with the plug type capillaryfitting. The female union has a 250 um stop with a through hole of 50 μmID between the emitter entrance and the plug type capillary fitting (seeFIG. 10 and table 2 for a description of the union and emitter). FIG. 11is an example of the assembled emitter and nano/capillary column.

TABLE 2 Details for FIG. 10 in Example 2. Element Number in FIG. 10Dimensions 100 Length: 0.25 mm 144 ID: 0.05 mm 120 10/32″ Threading,Length: 8 mm 180 PEEK ferrule 130 Fused Silica, ID: 15 μm, OD: 150 μm,Length: 30 mm 133 Ferrule Seat in Union, 8/32″ Threading 135 PEEKtubing, OD 1/32″, ID 180 μm, Length 26 mm

EXAMPLE 3 Assemble the Male Type Emitter Assembly with 20 μm ID Emitter

An integrated emitter assembly having the structure shown in FIG. 7 wasassembled, which has a male type end fitting. The emitter was made froma fused silica capillary with 20 μm ID and 150 μm OD. The emitter outletwas polished to a sharp tip. The emitter length is 47 mm The emitterinlet has a plug type capillary fitting. The emitter was protected bythe retractable sheath and the metal cartridge (See FIG. 12, which is aclose up view of part of the emitter assembly, and Table 3 for adescription of the union and emitter). FIG. 13 and FIG. 14 are examplesof the assembled emitter with a capillary and micro/analytical column,respectively.

TABLE 3 Details for FIG. 12 in Example 3. Element number in FIG. 12Dimensions 221 PEEK, OD: 1.15 mm, ID: 0.79 mm, Length: 6.3 mm 220Stainless Steel, OD: 1.6 mm, ID: 1.2 mm, Length: 6.3 mm 211 10/32″Threading, Length: 8 mm 177 PEEK ferrule 130 Fused Silica, ID: 20 μm,OD: 150 μm, Length: 47 mm 150 Ferrule Seat in Union, 8/32″ Threading 135PEEK tubing, OD 1/32″, ID 180 μm, Length 43 mm

The replaceable emitter assembly was assembled by first inserting theemitter into the protective PEEK sleeve. The plug type capillary fittingwas then attached to the PEEK sleeve. The emitter was then threadedthough the bolt and a ferrule was set on the opposite side of the plugfitting. The emitter was then threaded through the spring, slidingemitter protector and emitter casing. The casing is then attached to thebolt head by pressing on the pin points of the casing.

EXAMPLE 4 Assemble the Male Type Emitter Assembly with 30 μm ID Emitter

An integrated emitter assembly having the structure shown in FIG. 7 wasassembled, which has a male type end fitting. The emitter was made froma stainless steel capillary with 30 μm ID and 150 μm OD. The emitteroutlet was tapered with a sharp tip. The emitter length is 47 mm Theemitter inlet has a plug type capillary fitting and then the wholeemitter body was protected by the retractable sheath and the metalcartridge (See FIG. 12 and Table 4 for a description of the union andemitter). FIG. 13 and FIG. 14 are examples of the assembled emitter witha capillary and micro/analytical column, respectively.

TABLE 4 Details for FIG. 12 in Example 4. Element number in FIG. 12Dimensions 221 PEEK, OD: 1.15 mm, ID: 0.79 mm, Length: 6.3 mm 220Stainless Steel, OD: 1.6 mm, ID: 1.2 mm, Length: 6.3 mm 211 10/32″Threading, Length: 8 mm 177 PEEK ferrule 130 Stainless Steel, ID: 30 μm,OD: 150 μm, Length: 47 mm 150 Ferrule Seat in Union, 8/32″ Threading 135PEEK tubing, OD 1/32″, ID 180 μm, Length 43 mm

EXAMPLE 5 BSA Tryptic Digest Analysis using a 50 μm ID Column Couplingwith the Female Type Emitter Assembly Assembled in Example 1

To assess the function of the emitter assembly assembled in Example 1, ananoflow column, as shown in FIG. 11, was connected to the emitterassembly to analyze BSA tryptic digest.

The nano column was 50 μm ID and 15 cm long and was packed with PepMap™C18, 2 μm media (Thermo Fisher Scientific, Waltham, Mass., USA). Bothends of the nanoflow column had plug type capillary fittings designedfor convenience of use and low dead volume connection between theemitter and nano HPLC system. LTQ XL™ mass spectrometer (MS) (ThermoFisher Scientific, Waltham, Mass., USA) was used as the detector with anEASY-Spray™ ion source. The LC flow rate was set to 300 nL/min

The LC-MS running conditions include the following:

-   -   Nano HPLC system: UltiMate™ 3000 RSLCnano system (Thermo Fisher        Scientific, Waltham, Mass., USA) with a nano-flow selector    -   Flow rate: 300 nL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 15 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 1.9 kV

FIG. 15 shows a base peak chromatogram of the separation, and FIG. 16shows the extracted ion chromatogram of ion with m/z 722. The peakretention time is 14 min. The peak width at half height (PWHH) is 2.36seconds. The peak asymmetry factor (10% peak height) is 1.82.

EXAMPLE 6 BSA Tryptic Analysis using 75 μm ID Column Coupling with theFemale Type Emitter Assembly, Assembled in Example 1

A column with 75 μm ID and 15 cm long was also used to test the emitterassembly, assembled in Example 1. The column was packed with PepMap™C18, 2 μm media. Plug type capillary fittings were attached at both endsof the column for convenience and a low dead volume connection with theemitter and nano HPLC system. The column outlet was connected with theemitter assembly as shown in FIG. 11, which will be inserted in anEASY-Spray™ ion source mounted on a LTQ XL™ mass spectrometer for MSdetection. The column inlet connected to a nano HPLC instrument, and theflow rate was 300 nL/min.

The LC-MS running conditions include the following:

-   -   Nano HPLC system: UltiMate™3000 RSLCnano system with a nano-flow        selector    -   Flow rate: 300 nL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 15 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 1.9 kV

FIG. 17 shows the base peak chromatogram of the separation, and FIG. 18shows the extracted ion chromatogram of ion with m/z 722. The peakretention time is 12.82 min The peak width at half height (PWHH) is 2.53seconds. The peak asymmetry factor (10% peak height) is 1.43.

EXAMPLE 7 BSA Tryptic Digest Analysis using 150 μm ID Column Couplingwith the Female Type Emitter Assembly, Assembled in Example 2

To assess the function of the emitter assembly assembled in Example 2, acapillary column, as shown in FIG. 11, was connected to the emitterassembly to analyze BSA tryptic digest.

The capillary column was 150 μmID and 15 cm long. It was packed withPepMap™ C18, 2 μm media. Outside of both ends of the column, plug typecapillary fittings were created for convenience and low dead volumeconnection with the emitter and nano HPLC system. LTQ XL™ massspectrometer (MS) was used as the detector with an EASY-Spray™ ionsource. The flow rate was 1.2 μL/min.

The LC-MS running conditions include the following:

-   -   Nano HPLC system: UltiMate™ 3000 RSLCnano system with a        capillary-flow selector    -   Flow rate: 1.2 μL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 15 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 1.9 kV

FIG. 19 shows the base peak chromatogram of the separation, and FIG. 20shows the extracted ion chromatogram of ion with m/z 722. The peakretention time is 7.59 min The peak width at half height (PWHH) is 2.17seconds. The peak asymmetry factor (10% peak height) is 1.41.

EXAMPLE 8 BSA Tryptic Digest Analysis using 150 μm ID Column Couplingwith the Male Type Emitter Assembly, Assembled in Example 3

To assess the function of the emitter assembly assembled in Example 3, acapillary column, as shown in FIG. 13, was connected to the emitterassembly through a union to analyze BSA tryptic digest.

The capillary column was 150 μm ID and 15 cm long. It was packed withPepMap™ C18, 2 μm media. Plug type capillary fittings were created atthe column both ends for convenience and low dead volume connection withthe emitter and nano HPLC system, respectively. The column outletconnected with the emitter assembly through a metal union, which has a100 μm thick wall at the center and a 50 μm diameter through hole on thecenter of the wall. The emitter assembly will be plugged in anEASY-Spray™ ion source mounted on a LTQ XL™ mass spectrometer for ESI-MSdetection. The flow rate was 1.2 μL/min.

The LC-MS running conditions include the following:

-   -   Nano HPLC system: UltiMate™ 3000 RSLCnano system with a        capillary-flow selector    -   Flow rate: 1.2 μL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 15 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 1.9 kV

FIG. 21 shows the base peak chromatogram of the separation, and FIG. 22shows the extracted ion chromatogram of ion with m/z 722. The peakretention time is 6.86 min The peak width at half height (PWHH) is 3.06seconds. The peak asymmetry factor (10% peak height) is 1.13.

EXAMPLE 9 BSA Tryptic Digest Analysis using 250 μm ID Column with theMale Type Emitter Assembly, Assembled in Example 3

The male type emitter assembly assembled in Example 3 was investigatedby coupling it with a 250 μm ID capillary column for BSA tryptic digestanalysis.

The column was packed in a 250 μm ID 15 cm long fused silica capillaryand the packing media was PepMap™ C18, 2 μm. The whole column wasprotected in a PEEK sleeve and both ends of the column received a plugtype capillary fitting for convenient and low dead volume connection.Its inlet connected with a nano HPLC instrument. Its outlet wasconnected with the male type emitter assembly through a metal union (asshown in FIG. 13), which has a 100 μm thick wall at the center and a 50μm diameter through hole on the center of the wall. During testing, theemitter assembly was plugged into an EASY-Spray™ ion source, which wasmounted on a LTQ XL™ mass spectrometer, for ESI-MS detection. The flowrate was 3 μL/min.

The LC-MS running conditions include the following:

-   -   Nano HPLC system: UltiMate™ 3000 RSLCnano system with a        capillary-flow selector    -   Flow rate: 3 μL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 15 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 1.9 kV

FIG. 23 shows the base peak chromatogram of the separation, and FIG. 24shows the extracted ion chromatogram of ion with m/z 722. The peakretention time is 6.96 min The peak width at half height (PWHH) is 3.71seconds. The peak asymmetry factor (10% peak height) is 1.15.

EXAMPLE 10 BSA Tryptic Digest Analysis using 500 μm ID Column Couplingwith the Male Type Emitter Assembly, Assembled in Example 4

The male type emitter assembly assembled in Example 4 was tested using a500 μm ID micro column for BSA tryptic digest analysis.

The column was packed in a 500 μm ID and 10 cm long stainless steeltube, and packed with PepMap™ C18, 2 μm media. The emitter assemblycould be directly screwed into the column outlet end fitting and fingertightened as shown in FIG. 14. The emitter assembly was then insertedinto an EASY-Spray™ ion source, which was mounted on a LTQ XL™ massspectrometer, for ESI-MS detection. The column inlet was connected witha nano HPLC pump through a transfer tubing (50 μm ID) to provide liquidflow. The testing flow rate was set 12 μL/min

The LC-MS running conditions include the following:

-   -   Nano HPLC system: UltiMate™ 3000 RSLCnano system with a        micro-flow selector    -   Flow rate: 12 μL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 10 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 2.2 kV

FIG. 25 shows the base peak chromatogram of the separation, and FIG. 26shows the extracted ion chromatogram of ion with m/z 722. The peakretention time is 4.09 min The peak width at half height (PWHH) is 2.49seconds. The peak asymmetry factor (10% peak height) is 1.44.

EXAMPLE 11 Column Performance Comparison between the Emitter Assemblyused in Example 5 with Fully Integrated EASY-Spray™ Column (ES801,Thermo Fisher Scientific)

To evaluate the performance of the assembly used in Example 5, anEASY-Spray™ column with the same column format and emitter dimension(ES801, Thermo Fisher Scientific) was tested with BSA tryptic digestunder the same testing conditions as follows:

-   -   Nano HPLC system: UltiMate™ 3000 RSLCnano system with a        nano-flow selector    -   Flow rate: 300 nL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 15 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 1.9 kV

FIG. 27 shows the base peak chromatograms of the separation and theextracted ion chromatograms of ion with m/z 722 from both columns. Table5 list the peak retention times, the peak width at half heights (PWHH)and the peak asymmetry factors (10% peak height), which indicates bothcolumns provided comparable performance

TABLE 5 722 m/z Peak Performance. Peak Retention Width LCMS Column TypeTime (min) (sec) Asymmetry Emitter Assembly with 13.96 2.56 2.14 ColumnEASY-Spray ™ Column 13.60 2.60 2.19

EXAMPLE 12 Column Performance Comparison between the Emitter Assemblyused in Example 6 with Fully Integrated EASY-Spray™ Column (ES804,Thermo Fisher Scientific)

To evaluate the performance of the assembly used in Example 6, anEASY-Spray™ column with the same column format and emitter dimension(ES804, Thermo Fisher Scientific) was tested with BSA tryptic digestunder the same testing conditions as follows:

-   -   Nano HPLC system: UltiMate™ 3000 RSLCnano system with a        nano-flow selector    -   Flow rate: 300 nL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 15 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 1.9 kV

FIG. 28 shows the base peak chromatograms of the separation and theextracted ion chromatograms of ion with m/z 722 from both columns. Table6 lists the peak retention times, the peak width at half heights (PWHH)and the peak asymmetry factors (10% peak height), which indicates bothcolumns provided comparable performance

TABLE 6 722 m/z Peak Performance. Peak Retention Width LCMS Column TypeTime (min) (sec) Asymmetry Emitter Assembly with 12.43 2.53 1.60 ColumnEASY-Spray ™ Column 12.30 2.52 1.59

EXAMPLE 13 Column Performance Comparison between the Emitter Assemblyused in Example 7 with a Fully Integrated EASY-Spray™ Column (ES806,Thermo Fisher Scientific)

To evaluate the performance of the assembly used in Example 7, anEASY-Spray™ column with the same column format and emitter dimension(ES806, Thermo Fisher Scientific) was tested with BSA tryptic digestunder the same testing conditions as follows:

-   -   Nano HPLC system: UltiMate™ 3000 RSLCnano system with a        capillary-flow selector    -   Flow rate: 1.2 μL/min    -   Mobile phase A: 0.1% formic acid in water    -   Mobile phase B: 0.1% formic acid in acetonitrile    -   Gradient: 2-40% mobile phase B in 15 min, then 40-95% mobile        phase B in 5 min, and keep 95% mobile phase B for 5 min    -   Injection sample amount: 100 fmol BSA digest    -   Temperature: ambient    -   MS instrument: LTQ XL™ MS with EASY-Spray™ ion source    -   Spray voltage: 1.9 kV

FIG. 29 shows the base peak chromatograms of the separation and theextracted ion chromatograms of ion with m/z 722 from both columns. Table7 lists the peak retention times, the peak width at half heights (PWHH)and the peak asymmetry factors (10% peak height), which indicates bothcolumns provided comparable performance

TABLE 7 722 m/z Peak Performance. Peak Retention Width LCMS Column TypeTime (min) (sec) Asymmetry Emitter Assembly with 7.67 2.68 1.21 ColumnEASY-Spray ™ Column 7.47 2.47 1.24

The present invention has been described in terms of specificembodiments incorporating details to facilitate the understanding of theprinciples of construction and operation of the invention. As such,references herein to specific embodiments and details thereof are notintended to limit the scope of the claims appended hereto. It will beapparent to those skilled in the art that modifications can be made inthe embodiments chosen for illustration without departing from thespirit and scope of the invention.

1. (canceled)
 2. An electrospray emitter assembly for interfacing aseparation column to a mass spectrometer, comprising: a. an emittercapillary having an inlet end and an outlet end; and b. a fittingcoupled to the inlet end of the emitter, configured to be removablyconnected to the separation column; wherein a stop with a definedthrough hole is integrated proximate the inlet end of the emitter toproduce a path for liquid to flow from the separation column to theemitter via the through hole where a voltage is applied to the liquidentering the emitter.
 3. The emitter assembly of claim 2 wherein thefitting is a female threaded end fitting.
 4. The emitter assembly ofclaim 2 wherein the emitter capillary comprises a fused silicacapillary, a metal capillary, a ceramic capillary, or a glass capillary.5. The emitter assembly of claim 2 wherein the separation column isremovably connected via a transfer line with the emitter capillarythrough the fitting.
 6. The emitter assembly of claim 2 wherein theintegrated stop has a thickness of up to 1.0 mm.
 7. The emitter assemblyof claim 2 wherein the integrated stop has a thickness of between 100 μmto 300 μm.
 8. The emitter assembly of claim 2 wherein the through holein the stop has a diameter of between 3μm and 100 μm.
 9. The emitterassembly of claim 2 further comprising a protective sleeve for coveringand supporting the emitter.
 10. The emitter assembly of claim 9 whereinthe protective sleeve is slidably mounted around the emitter.
 11. Theemitter assembly of claim 10 further comprising an electricallyconductive outer sheath, wherein the sleeve is at least partiallyenclosed and moveable within the outer sheath.
 12. An electrosprayemitter assembly for interfacing a separation column to a massspectrometer, comprising: a. an emitter capillary having an inlet endand an outlet end; and b. a plug type end fitting coupled to the inletend of the emitter, configured to be removably connected to theseparation column.
 13. The emitter assembly of claim 12 wherein theemitter capillary comprises a fused silica capillary, a metal capillary,a ceramic capillary, or a glass capillary.
 14. The emitter assembly ofclaim 12 wherein the separation column is removably connected via atransfer line with the emitter capillary through the plug type endfitting.
 15. The emitter assembly of claim 12 wherein the plug type endfitting is a male threaded end fitting.
 16. The emitter assembly ofclaim 12 further comprising a protective sleeve for covering andsupporting the emitter.
 17. The emitter assembly of claim 16 wherein theprotective sleeve is slidably mounted around the emitter.
 18. Theemitter assembly of claim 17 further comprising an electricallyconductive outer sheath, wherein the protective sleeve is at leastpartially enclosed and moveable within the outer sheath.
 19. A methodfor analyzing a sample, comprising: threading a fitting coupled to aseparation column into a threaded inlet of a electrospray emitterassembly, the electrospray emitter assembly including an emittercapillary having an inlet end and an outlet end, a union coupled to theinlet end of the emitter capillary, and a protective sleeve slidablymounted around the emitter capillary for covering and supporting theemitter capillary, the union including a defined through hole integratedproximate the inlet end of the emitter capillary, wherein a low deadvolume flow path is established from the separation column to theemitter capillary through the defined through hole, wherein the fittingcouples directly with the union and the union couples directly to theemitter capillary; inserting the electrospray emitter assembly into aholder, wherein an electrically conductive sheath of the electrosprayemitter assembly engages a high voltage contact of the holder therebyestablishing an electrical path from the holder through the electricallyconductive sheath to the union, wherein the protective sleeve is atleast partially enclosed and movable within the electrically conductivesheath; moving the protective sleeve to expose the outlet end of theemitter capillary; flowing a sample from the separation column throughthe union to the emitter capillary; applying an electrical currentthrough the electrically conductive sheath of the electrospray emitterassembly such that the current is applied to the liquid passing throughthe union, electrospraying droplets from the outlet end of the emittercapillary to a mass spectrometer; and analyzing, in the massspectrometer, ions produced during the electrospraying.